Optical architectures for microvolume laser-scanning cytometers

Details Optical architectures for microvolume laser-scanning cytometers Optical architectures for microvolume laser-scanning cytometers

2003-08-05

2004-10-05

Gagliardi, Albert (Department: 2878)

Radiant energy

Luminophor irradiation

C250S459100, C356S317000, C356S318000

Reexamination Certificate

active

06800860

ABSTRACT:

BACKGROUND OF THE INVENTION
Microvolume laser scanning cytometry (MLSC) is a method for analyzing the expression of biological markers in a biological fluid. See, e.g., U.S. Pat. Nos. 5,547,849 and 5,556,764; Dietz et al., Cytometry 23:177-186 (1996); U.S. Provisional Application No. 60/144,798, filed Jul. 21, 1999, each of which is incorporated herein by reference. A sample, such as blood, is incubated in a capillary with one or more fluorescently-labeled probes that specifically binds to particular biological markers, such as membrane proteins displayed on the surface of a blood cell. The sample is then analyzed by a MLSC instrument, which scans excitation light from a laser over the sample along one axis of the capillary, while the capillary itself is moved in an orthogonal axis by an automated stage. Fluorescent probes in the sample emit Stokes-shifted light in response to the excitation light, and this light is collected by the cytometer and used to form an image of the sample. In such images, the cells or particles that bind to the fluorescent probes can be identified and quantitated by image analysis algorithms. The resulting information on the expression of biological markers in the sample can be used for diagnostic and prognostic medical purposes.
Current laser scanning cytometers are based on the flying spot confocal laser scanner. These systems scan a laser excitation light spot in one dimension across the sample using a rotating or reciprocating mirror, such as a mirror mounted on a galvanometer. The sample is translated in a direction orthogonal to the scan direction. The collimated excitation laser light follows an epi-illumination path through the microscope objective and is focused on the sample and the mirror scan center is imaged upon the entrance pupil of the microscope objective. Emitted light from the sample is then collected by the microscope objective, and re-traces the excitation light path back to the scanning mirror where it is descanned. The emitted light passes through a dichroic filter and a long-pass filter to separate out reflected excitation light, and is then focussed onto an optical detector through an aperture. The aperture serves as a spatial filter, and reduces the amount of out-of-focus light that is introduced into the detector. The wider the aperture, the greater the depth of focus of the system. The detector generates a signal that is proportional to the intensity of the incident light. Thus, as the laser scans the sample, an image is assembled pixel-by-pixel. This optical architecture is typically referred to as confocal fluorescence detection.
In order to detect multiple fluorescence probes, the laser scanning cytometry system can also include dichroic filters that separate the emitted light into its component wavelengths. Each distinct wavelength is imaged onto a separate detector through a separate aperture. In this way, an image of the sample is assembled pixel-by-pixel for each emitted wavelength. The individual images are termed channels, and the final multi-color image is obtained by merging the individual channels.
The use of confocal 4-channel fluorescence detection for MLSC is illustrated schematically in
FIG. 1
, and is described in the U.S. Provisional Patent Application entitled “Improved System for Microvolume Laser Scanning Cytometry”, filed Jul. 21, 1999, incorporated herein by reference in its entirety. In this embodiment, the light from a laser is scanned over a capillary array
8
wherein each capillary contains a sample that contains fluorescently-labeled species. Specifically, collimated excitation light is provided by a He—Ne laser
10
. The collimated laser light is deflected by an excitation dichroic filter
12
. Upon reflection, the light is incident on a galvanometer-driven scan mirror
14
. The scan mirror can be rapidly oscillated over a fixed range of angles by the galvanometer e.g. ±2.5 degrees. The scanning mirror reflects the incident light into two relay lenses (relay lens
1
(
16
) and relay lens
2
(
18
)) that image the scan mirror onto the entrance pupil of the microscope objective
20
. This optical configuration converts a specific scanned angle at the mirror to a specific field position at the focus of the microscope objective
20
. The angular sweep is typically chosen to result in a 1 mm scan width at the objective's focus. The relationship between the scan angle and the field position is essentially linear in this configuration and over this range of angles. Furthermore the microscope objective
20
focuses the incoming collimated beam to a spot at the objective's focus plane. The spot diameter, which sets the optical resolution, is determined by the diameter of the collimated beam and the focal length of the objective. Fluorescence samples placed in a capillary array
8
in the path of the swept excitation beam emit stokes-shifted light. This light is collected by the objective
20
and collimated. This collimated light emerges from the two relay lenses (
16
and
18
) still collimated and impinges upon the scan mirror
14
which reflects and descans it. The stokes-shifted light then passes through an excitation dichroic filter
12
(most excitation light reflected within the optics to this point is reflected by this dichroic filter) and then through long pass filter
1
(
22
) that further serves to filter out any reflected excitation light. Fluorescence dichroic filter
1
(
24
) then divides the two bluest fluorescence colors from the two reddest. The two bluest colors are then focussed through focusing lens
1
(
26
) onto aperture
1
(
28
) in order to significantly reduce any out-of-focus fluorescence signal. After passing though the aperture, fluorescence dichroic
2
(
30
) further separates the individual blue colors from one another. The individual blue colors are then parsed to two separate photomultipliers
1
(
32
) and
2
(
34
). After being divided from the two bluest colors by fluorescence dichroic
1
(
24
), the two reddest colors are passed through long pass filter
2
(
36
) and reflected off a mirror (
38
) through focusing lens
2
(
40
) onto aperture
42
. After passing through aperture
2
, the reddest colors are separated from one another by fluorescence dichroic
3
(
44
). The individual red colors are then parsed to photomultipliers
3
(
46
) and
4
(
48
). In this way, four separate fluorescence signals can be simultaneously transmitted from the sample held in the capillary to individual photomultiplier light detectors (PMT
1
-
4
). Each photomultiplier generates an electronic current in response to the incoming fluorescence photon flux. These individual currents are converted to separate voltages by one or more preamplifiers in the detection electronics. The voltages are sampled at regular intervals by an analog to digitial converter in order to determine pixel intensity values for the scanned image.
Other ways are known in the art for obtaining multi-channel information in the microscopy context. For example, it is known in the art to use fluorophores that emit light with overlapping emission spectra but with different time constants of emission. Time-resolved microscopy systems typically use very fast laser pulses and high speed detection circuitry to resolve, in the time-domain, the nanosecond-scale time signatures of the fluorophores. Alternatively, the measurement can be accomplished in the frequency domain with amplitude-modulated laser sources and detection circuitry that measures phase shift and modulation amplitudes. Both of these techniques add significant complexity to the fluorescent measurement system.
Typical MLSC instruments use photomultiplier tubes (PMTs) as light detectors. PMTs are cost-effective and have high data read-out rates which allow the sample to be scanned swiftly. However, a major drawback of the PMT is its low quantum efficiency. For example, in the red to near infrared region of the electromagnetic spectrum, PMTs have quantum efficiencies of less than 10%, i.e., less than one photon in ten that impacts the

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